The objective of this application is to understand how opsin is synthesized into endoplasmic reticulum (ER) membranes. The specific aims are to examine how the amino terminus of opsin crosses the membrane and how opsin integrates into the lipid bilayer. Each of these questions will be addressed both to wild-type opsin as well as some of its point mutants described in autosomal dominant retinitis pigmentosa (RP). Studying the pathologies in these mutants' biogenesis may yield insights into normal physiological pathways of opsin synthesis. Understanding the consequences of these mutations on the biogenesis of opsin may also contribute to our understanding of the pathogenesis of RP. Many of these mutations involve substitutions in regions of the opsin molecule that are critical to its biogenesis. Opsin biogenesis will be studied by trapping incremental stages of synthesis and quantifying: opsin targeting to the ER; translocation across the ER; folding of opsin; interaction with the translocation protein-conducting channels; interactions with ER resident proteins; and integration into the membrane. This application takes a multidisciplinary approach that combines using (1) truncated mRNA to study intermediates in biogenesis; (2) electrophysiology to study the protein-conducting channels; (3) purified reconstituted components of the ER to examine the interactions of opsin with the translocation machinery; (4) photo-activated cross-linkers in nascent opsin to identify ER components interacting with nascent opsin. This work is relevant to the larger class of clinically important polytopic membrane proteins, the G-protein coupled receptors, which share many structural features with opsin. Finally, identification and characterization of the topogenic signals within opsin itself, and a description of how they interact with the translocation machinery, provides a paradigm for understanding general membrane protein biogenesis.